Acute lung Injury/Acute Respiratory Distress Syndrome (ALI/ARDS) is a common cause of respiratory failure in critically ill patients, and afflicts ~200,000 Americans each year with ~75000 deaths and 3.6 million hospital days. The hallmark of ALI is vascular dysfunction characterized by endothelial cell (EC) inflammation and barrier disruption resulting in inflammatory infiltrates, interstitial edema, alveolar flooding, and ultimately respiratory failure. All current therapies for ALI/ARDS rely on supportive care to improve clinical outcome. No effective drugs have been developed. Thus, there is an urgent need to develop new treatment strategies for ALI/ARDS that are safe, effective, and based on deeper understanding of the mechanisms involved in ALI pathogenesis. Our long-term goal is to identify viable therapeutic targets and mechanisms to limit ALI. The objective of this application is to determine the role of ER chaperone BiP (immunoglobulin heavy chain binding protein) and mitochondrial chaperone mortalin in mediating EC barrier dysfunction and inflammation in ALI and assess the therapeutic benefit of targeting these molecules against ALI. The proposal is based on our novel findings that implicate an important role for BiP and mortalin in mediating inflammatory signaling and barrier disruption in EC and lung PMN infiltration and vascular leak in a mouse model of ALI. Our encouraging data show that pharmacological inhibitor of BiP or mortalin each protects against lung injury. Intriguingly, however, the combined inhibition of BiP and mortalin is effective at much lower dosage of each inhibitor (which alone shows no protective effect) in protecting against LPS-induced lung injury and mortality in mice. These exciting findings have led us to the hypothesis that BiP and mortalin are critical determinants of ALI by their ability to promote EC permeability and inflammation, and that combined inhibition of BiP and mortalin may prove a highly efficacious therapeutic intervention to control ALI. The proposal will address the following aims. Aim 1 will determine (i) the mechanism of BiP regulation of EC permeability and inflammation and (ii) in vivo role of endothelial BiP in causing lung inflammation and injury. Aim 2 will determine (i) the mechanism by which mortalin regulates EC permeability and inflammation and (ii) in vivo role of endothelial mortalin in causing lung inflammation and injury. Aim 3 will evaluate the preventive and therapeutic potential of simultaneous targeting (combined inhibition) of BiP and mortalin against ALI and mortality in mice. These studies will utilize a combination of cellular, molecular, biochemical, pharmacological, imaging, in vivo gene transfer, and lung physiology. The creative integration of in vitro and in vivo studies will provide novel insights into BiP and mortalin regulation of EC inflammation and permeability during ALI and may lead to novel therapeutic interventions to control ALI/ARDS.